Glucagon/GLP-1 agonists for the treatment of obesity

ABSTRACT

This disclosure provides GLP-1/glue agon agonist peptides for the treatment of metabolic diseases, e.g., obesity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application of InternationalApplication No. PCT/IB2013/003191, filed on Dec. 10, 2013, saidInternational Application No. PCT/IB2013/003191 claims benefit under 35U.S.C. §119(e) of the U.S. Provisional Application No. 61/735,823, filedDec. 11, 2012. Each of the above listed applications is incorporated byreference herein in its entirety for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: sequencelisting_ascii.txt; Size: 12.3 kilobytes; andDate of Creation: Dec. 10, 2013) filed with the application isincorporated herein by reference in its entirety.

BACKGROUND

Obesity is a major and growing health problem worldwide, and isassociated with many life-threatening diseases such as cardiovasculardisease, renal disease, hypertension, stroke, infertility, respiratorydysfunction, and type 2 diabetes.

Glucagon and glucagon-like peptide-1 (GLP-1) derive frompre-proglucagon, a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofproglucagon (53 to 81 of preproglucagon), while GLP-1 is produced as a37-amino acid peptide that corresponds to amino acids 72 through 108 ofproglucagon (92 to 128 of preproglucagon). GLP-1(7-36) amide orGLP-1(7-37) acid are biologically active forms of GLP-1, thatdemonstrate essentially equivalent activity at the GLP-1 receptor.

Glucagon is produced by the pancreas and interacts with the glucagonreceptor (“glucR”). Glucagon acts in the liver to raise blood glucosevia gluconeogenesis and glycogenolysis. When blood glucose begins tofall, glucagon signals the liver to break down glycogen and releaseglucose, causing blood glucose levels to rise toward a normal level.

GLP-1 has different biological activities compared to glucagon. It issecreted from gut L cells and binds to the GLP-1 receptor. Itsactivities include stimulation of insulin synthesis and secretion,inhibition of glucagon secretion, and inhibition of food intake.

Both glucagon and GLP-1, acting as agonists at their respectivereceptors, have been shown to be effective in weight loss. Certain GLP-1analogs are being sold or are in development for treatment of obesityincluding, e.g., Liraglutide (VICTOZA® from Novo Nordisk) and Exenatide(Byetta® from Eli Lilly/Amylin).

There remains a need for more agents for effective treatment of obesity,for example, GLP-1/Glucagon agonist peptides with improved solubility,formulatability, stability, and efficacy.

BRIEF SUMMARY

This disclosure provides an isolated peptide comprising or consisting ofthe amino acid sequence:

-   -   HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X 24WLX27X28GX30;        where X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is        K or Y, X15 is D or E. X16 is S or G, X17 is E, R, Q, or K, X18        is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I,        X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R        (SEQ ID NO: 4). In certain aspects, X2 is S, X15 is D, X16 is S,        X20 is R, X21 is D, X23 is V, X24 is A, X28 is A, and X30 is G        (SEQ ID NO:5). In certain aspects, if X17 is E, then X18 is R,        and if X17 is R, then X18 is S (SEQ ID NOs:6 and 7). In certain        aspects, X10 is Y, X12 is K, X13 is K, and X27 is V (SEQ ID        NOs:8 and 9). In certain aspects, X10 is K, X13 is Y, and X27 is        E (SEQ ID NOs:10 and 11). In certain aspects, X12 is E (SEQ ID        NOs:12 and 13), alternatively, X12 is R (SEQ ID NOs:14 and 15).        In certain aspects, the isolated peptide comprises, or consists        of SEQ ID NO:16. In certain aspects, the isolated peptide        comprises, or consists of the amino acid sequence SEQ ID NOs:17        or the amino acid sequence SEQ ID NO:19. In certain aspects, the        isolated peptide comprises, or consists of SEQ ID NO:18.

In certain embodiments of the peptides described above, the carboxylgroup of X30 is amidated. In other embodiments the carboxyl group is theunmodified acid.

Any of the peptides provided herein can further comprise one or moremodified amino acids, for example, the addition of an acyl moiety, forexample, the modification can be the addition of a palmitoyl moiety onthe N(epsilon) group of a lysine residue. In certain embodiments, thepalmitoyl group is linked to the lysine residue through a gammaglutamate linker. Alternative linkers have been used including betaalanine and aminohexanoic acid. Further alternative linkers are possibleincluding linkers containing short PEG moieties for instance containing2 or 4 PEG units.

In various embodiments, the isolated peptides provided herein can bindto a glucagon receptor, to a GLP-1 receptor, or to both a glucagon and aGLP-1 receptor. In certain aspects the glucagon receptor is a humanglucagon receptor, and or the GLP-1 receptor is a human GLP-1 receptor.In certain aspects an isolated peptide as provided herein binds to ahuman glucagon receptor with an EC50 in the cAMP assay 1 (as describedherein) of less than 10,000 pM, less than 5000 pM, less than 2500 pM,less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM,less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM,less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM,less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, lessthan 4 pM, less than 3 pM, or less than 2 pM. In certain aspects anisolated peptide as provided herein binds to a human GLP-1 receptor withan EC50 in the cAMP assay 1 of less than 10,000 pM, less than 5000 pM,less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM,less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

In certain aspects, an isolated peptide as provided herein is an agonistof GLP-1 activity, an agonist of glucagon activity, or an agonist ofboth GLP-1 and glucagon activity. In some embodiments, an isolatedpeptide as provided herein binds to both a glucagon receptor and a GLP-1receptor, and exhibits at least about 2-fold greater activity relativeto the natural ligand at the GLP-1 receptor than at the glucagonreceptor. In one embodiment the peptide has a 5 to 10 fold higherrelative potency at the GLP1R, compared to GLP1, than at the glucagonreceptor, relative to glucagon.

In certain aspects, an isolated peptide as provided herein can furthercomprise a heterologous moiety associated with the peptide. In someaspects, the heterologous moiety is a protein, a peptide, a proteindomain, a linker, an organic polymer, an inorganic polymer, apolyethylene glycol (PEG), biotin, an albumin, a human serum albumin(HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody,an antibody fragment, a single chain antibody, a domain antibody, analbumin binding domain, an enzyme, a ligand, a receptor, a bindingpeptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptidepolymer, a cytokine, or any combination of two or more of such moieties.

Also provided is a pharmaceutical composition comprising an isolatedpeptide as described herein, and a carrier. Further provided is a kitincluding such a pharmaceutical composition.

Also provided is a method for treating or preventing a disease orcondition caused or characterized by excess body weight, where themethod includes administering to a subject in need of treatment aneffective amount of an isolated peptide as provided herein, or acomposition which includes such a peptide. In certain aspects, thedisease or condition can be obesity, insulin resistance, glucoseintolerance, pre-diabetes, increased fasting glucose, type 2 diabetes,hypertension, dyslipidemia (or a combination of these metabolic riskfactors), glucagonomas, cardiovascular disease, e.g., congestive heartfailure, atherosclerois, arteriosclerosis, coronary heart disease, orperipheral artery disease; stroke, respiratory dysfunction, renaldisease, and any combination thereof. According to the method, anisolated peptide as described herein can be administered by injection,e.g., subcutaneous injection. According to the method, the peptide canbe administered once per day. In certain embodiments, the subject is ahuman.

Also provided is a method for treating or preventing a disease orcondition caused or characterized by excess body weight, where themethod includes administering to a subject in need of treatment aneffective amount of an isolated peptide as provided herein, or acomposition which includes such a peptide. According to the method, anisolated peptide as described herein can be administered by injection,e.g., subcutaneous injection. According to the method, the peptide canbe administered once per day. In certain embodiments, the subject is ahuman.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG730 at three different doses, compared to vehicle treatment, andtreatment with Liraglutide. Starting body weight in the different groupswere vehicle: 47.4±3.7 g, G730 10 nmol/kg: 44.5±2.2 g, G730 20 nmol/kg:45.9±3.6 g and G730 50 nmol/kg: 46.1±2.4 g, respectively.

FIG. 2 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG797 at three different doses, compared to vehicle treatment, andtreatment with Liraglutide. Starting body weight in the different groupswere vehicle: 47.4±3.7 g, G797 5 nmol/kg: 47.5±1.2 g, G797 20 nmol/kg:47.4±2.2 g and G797 50 nmol/kg: 47.2±1.8 g, respectively.

FIG. 3 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG812 at a dose of 20 nmol/kg, compared to vehicle treatment, andtreatment with Liraglutide. Starting body weight in the different groupswere vehicle: 47.4±3.7 g and G812 20 nmol/kg: 49.2±3.4 g, respectively.

FIG. 4 is a graph comparing the change in body weight results for thethree glucagon/GLP-1 co-agonist peptides presented in FIGS. 1, 2, and 3.

FIG. 5 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG796 at two different doses, compared to vehicle treatment, andtreatment with Liraglutide.

FIG. 6 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG865 at two different doses, compared to vehicle treatment, andtreatment with Liraglutide.

FIG. 7 shows the mean percent of change in body weight from day zero inDIO mice following administration of glucagon/GLP-1 co-agonist peptideG933 at two different doses, compared to vehicle treatment, andtreatment with Liraglutide.

FIG. 8 is a graph comparing the change in body weight results for thethree glucagon/GLP-1 co-agonist peptides presented in FIGS. 5, 6, and 7.

DETAILED DESCRIPTION Definitions

Throughout this disclosure, the term “a” or “an” entity refers to one ormore of that entity; for example, “a polynucleotide,” is understood torepresent one or more polynucleotides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein, a“peptide,” a “peptide subunit,” a “protein,” an “amino acid chain,” an“amino acid sequence,” or any other term used to refer to a chain orchains of two or more amino acids, are included in the definition of a“polypeptide,” even though each of these terms can have a more specificmeaning. The term “polypeptide” can be used instead of, orinterchangeably with any of these terms. The term further includespolypeptides which have undergone post-translational or post-synthesismodifications, for example, glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, or modification by non-naturally occurring aminoacids.

More specifically, the term “peptide” as used herein encompasses a fulllength peptides and fragments, variants or derivatives thereof, e.g., aGLP-1/glucagon agonist peptide (e.g., 29, 30, or 31 amino acids inlength). A “peptide” as disclosed herein, e.g., a GLP-1/glucagon agonistpeptide, can be part of a fusion polypeptide comprising additionalcomponents such as, e.g., an Fc domain or an albumin domain, to increasehalf-life. A peptide as described herein can also be derivatized in anumber of different ways.

The terms “fragment,” “analog,” “derivative,” or “variant” whenreferring to a GLP-1/glucagon agonist peptide includes any peptide whichretains at least some desirable activity, e.g., binding to glucagonand/or GLP-1 receptors. Fragments of GLP-1/glucagon agonist peptidesprovided herein include proteolytic fragments, deletion fragments whichexhibit desirable properties during expression, purification, and oradministration to an subject.

The term “variant,” as used herein, refers to a peptide that differsfrom the recited peptide due to amino acid substitutions, deletions,insertions, and/or modifications. Variants can be produced usingart-known mutagenesis techniques. Variants can also, or alternatively,contain other modifications—for example a peptide can be conjugated orcoupled, e.g., fused to a heterologous amino acid sequence or othermoiety, e.g., for increasing half-life, solubility, or stability.Examples of moieties to be conjugated or coupled to a peptide providedherein include, but are not limited to, albumin, an immunoglobulin Fcregion, polyethylene glycol (PEG), and the like. The peptide can also beconjugated or produced coupled to a linker or other sequence for ease ofsynthesis, purification or identification of the peptide (e.g., 6-His),or to enhance binding of the polypeptide to a solid support.

The term “sequence identity” as used herein refers to a relationshipbetween two or more polynucleotide sequences or between two or morepolypeptide sequences. When a position in one sequence is occupied bythe same nucleic acid base or amino acid in the corresponding positionof the comparator sequence, the sequences are said to be “identical” atthat position. The percentage “sequence identity” is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid occurs in both sequences to yield the number of“identical” positions. The number of “identical” positions is thendivided by the total number of positions in the comparison window andmultiplied by 100 to yield the percentage of “sequence identity.”Percentage of “sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window. In order tooptimally align sequences for comparison, the portion of apolynucleotide or polypeptide sequence in the comparison window cancomprise additions or deletions termed gaps while the reference sequenceis kept constant. An optimal alignment is that alignment which, evenwith gaps, produces the greatest possible number of “identical”positions between the reference and comparator sequences. Percentage“sequence identity” between two sequences can be determined using theversion of the program “BLAST 2 Sequences” which was available from theNational Center for Biotechnology Information as of Sep. 1, 2004, whichprogram incorporates the programs BLASTN (for nucleotide sequencecomparison) and BLASTP (for polypeptide sequence comparison), whichprograms are based on the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing “BLAST 2Sequences,” parameters that were default parameters as of Sep. 1, 2004,can be used for word size (3), open gap penalty (11), extension gappenalty (1), gap drop-off (50), expect value (10), and any otherrequired parameter including but not limited to matrix option.

The terms “composition” or “pharmaceutical composition” refer tocompositions containing a GLP-1/glucagon agonist peptide providedherein, along with e.g., pharmaceutically acceptable carriers,excipients, or diluents for administration to a subject in need oftreatment, e.g., a human subject being treated for obesity.

The term “pharmaceutically acceptable” refers to compositions that are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity orother complications commensurate with a reasonable benefit/risk ratio.

An “effective amount” is that amount of a GLP-1/glucagon agonist peptideprovided herein, the administration of which to a subject, either in asingle dose or as part of a series, is effective for treatment, e.g.,treatment of obesity. An amount is effective, for example, when itsadministration results in one or more of weight loss or weightmaintenance (e.g., prevention of weight gain), loss of body fat,prevention or modulation hypoglycemia, prevention or modulationhyperglycemia, promotion of insulin synthesis, or reduction in foodintake. This amount can be a fixed dose for all subjects being treated,or can vary depending upon the weight, health, and physical condition ofthe subject to be treated, the extent of weight loss or weightmaintenance desired, the formulation of peptide, a professionalassessment of the medical situation, and other relevant factors.

The term “subject” is meant any subject, particularly a mammaliansubject, in need of treatment with a GLP-1/glucagon agonist peptideprovided herein. Mammalian subjects include, but are not limited to,humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle,bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on. Inone embodiment, the subject is a human subject.

As used herein, an “subject in need thereof” refers to an individual forwhom it is desirable to treat, e.g., to an obese subject or a subjectprone to obesity for whom it is desirable to facilitate weight or bodyfat loss, weight or body fat maintenance, or to prevent or minimizeweight gain over a specified period of time.

As used herein a “GLP-1/glucagon agonist peptide” is a chimeric peptidethat exhibits activity at the glucagon receptor of at least about 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relativeto native glucagon and also exhibits activity at the GLP-1 receptor ofabout at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or more relative to native GLP-1, under the conditions ofassay 1.

As used herein the term “native glucagon” refers to naturally-occurringglucagon, e.g., human glucagon, comprising the sequence of SEQ ID NO: 1.The term “native GLP-1” refers to naturally-occurring GLP-1, e.g., humanGLP-1, and is a generic term that encompasses, e.g., GLP-1(7-36) amide(SEQ ID NO: 2), GLP-1(7-37) acid (SEQ ID NO: 3) or a mixture of thosetwo compounds. As used herein, a general reference to “glucagon” or“GLP-1” in the absence of any further designation is intended to meannative human glucagon or native human GLP-1, respectively. Unlessotherwise indicated, “glucagon” refers to human glucagon, and “GLP-1”refers to human GLP-1.

GLP-1/Glucagon Agonist Peptides

Provided herein are peptides which bind both to a glucagon receptor andto a GLP-1 receptor. In certain embodiments, the peptides providedherein are co-agonists of glucagon and GLP-1 activity. Such peptides arereferred to herein as GLP-1/glucagon agonist peptides. GLP-1/glucagonagonist peptides as provided herein possess GLP-1 and glucagonactivities with favorable ratios to promote weight loss, prevent weightgain, or to maintain a desirable body weight, and possess optimizedsolubility, formulatability, and stability. In certain embodiments,GLP-1/glucagon agonist peptides as provided herein are active at thehuman GLP1 and human glucagon receptors, in certain embodiment relativeactivity compared to the natural ligand at the GLP-1 receptor is atleast about 1-fold, 2-fold 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, or25-fold higher than at the glucagon receptor.

In certain embodiments, GLP-1/glucagon agonist peptides as disclosedhave desirable potencies at the glucagon and GLP-1 receptors, and havedesirable relative potencies for promoting weight loss. In certainembodiments, GLP-1/glucagon agonist peptides as disclosed exhibit invitro potencies at the GLP-1 receptor as shown by an EC50 in the cAMPassay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM, lessthan 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM,less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM,less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM,less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, lessthan 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certainembodiments, GLP-1/glucagon agonist peptides as disclosed exhibit invitro potencies at the GLP-1 receptor as shown by EC50 in the cAMP assayin 4.4% human serum albumin (assay 2, see Example 2) of less than 10,000pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, orless than 2 pM. In certain embodiments, GLP-1/glucagon agonist peptidesas disclosed exhibit in vitro potencies at the glucagon receptor asshown by an EC50 in the cAMP assay 1 (see Example 2) of less than 10,000pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, orless than 2 pM. In certain embodiments, GLP-1/glucagon agonist peptidesas disclosed exhibit in vitro potencies at the glucagon receptor asshown by an EC50 in the cAMP assay in 4.4% human serum albumin (assay 2,see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM,less than 4 pM, less than 3 pM, or less than 2 pM. In certainembodiments, GLP-1/glucagon agonist peptides as disclosed have relativeGLP1-R/glucR potency ratios, when compared to the native ligands, in therange of about 0.01 to 0.50, e.g., from about 0.02 to 0.30, e.g., about0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11. 0.12, 0.13,0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25,0.26, 0.27, 0.28, or 0.30. when using assay 2.

In certain embodiments, GLP-1/glucagon agonist peptides as disclosedexhibit in vitro potencies at the glucose-dependent insulinotropicpeptide (gastric inhibitory peptide) (GIPR) as shown by an EC50 in thecAMP assay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM,less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM,less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. Incertain embodiments, GLP-1/glucagon agonist peptides as disclosedexhibit in vitro potencies at the GIPR as shown by EC50 in the cAMPassay in 4.4% human serum albumin (assay 2, see Example 2) of less than10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, lessthan 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, lessthan 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, lessthan 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, lessthan 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3pM, or less than 2 pM.

In certain embodiments, GLP-1/glucagon agonist peptides provided hereinpossess one or more criteria of acceptable solubility, ease informulatability, plasma stability, and improved pharmacokineticproperties. In certain embodiments, GLP-1/glucagon agonist peptides asdisclosed are soluble in standard buffers over a broad pH range.

In certain embodiments, GLP-1/glucagon agonist peptides are soluble incommon buffer solutions at a concentration up to 0.5 mg/ml, 0.6 mg/ml,0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, or more, in buffersystems and a range of ionic strengths, e.g., from 0.25 to 150 mM,including, but not limited to phosphate buffer, Tris buffer, glutamatebuffer, acetate buffer, succinate buffer, or histidine buffer. Exemplarybuffers include 100 mM glutamate pH 4.5 buffer, 100 mM acetate pH 5buffer, 100 mM succinate pH 5 buffer, 100 mM phosphate pH 6 buffer, 100mM histidine pH 6 buffer, 100 mM phosphate pH 6.5 buffer, 100 mMphosphate pH 7.0 buffer, 100 mM histidine pH 7.0 buffer, 100 mMphosphate pH 7.5 buffer, 100 mM Tris pH 7.5 buffer, and 100 mM Tris pH8.0 buffer. In certain embodiments, GLP-1/glucagon agonist peptides asdisclosed are soluble in standard buffers at 0.8 mg/ml over a range ofpH, e.g., from pH 4.0 to pH 8.0, e.g., at pH 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, or 8.5. In certain embodiments, GLP-1/glucagonagonist peptides as disclosed are soluble in standard buffers from pH4.5 to 8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0,4.5 to 8.5, 5.5 to 8.5, 5.5 to 8.5, 6.0 to 8.5, 6.5 to 8.5, or 7.0 to8.5.

In certain embodiments, GLP-1/glucagon agonist peptides as disclosed areformulatable in standard pharmaceutical formulations. Exemplaryformulations include, but are not limited to: 0.1M Tris pH 7.5, 150 mMMannitol, final formulation pH=7.2; 0.05M Tris, 50 mM Arginine/Proline,final formulation pH=8.0; or sodium phosphate buffer (pH8)/1.85% W/Vpropylene glycol, final formulation pH=7.0. In certain embodimentsGLP-1/glucagon agonist peptides as disclosed are soluble is these orother formulations at a concentration up to 0.5 mg/ml, 0.6 mg/ml, 0.7mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, or more.

In certain embodiments, GLP-1/glucagon agonist peptides as disclosed areacceptably stable against proteases in serum or plasma. Commondegradation products of glucagon or GLP-1 include +1 products (acid) andthe DPP IV-cleavage products. Products with +1 mass may arise fromdeamidation at amide groups of glutamine or at the C-terminus Cleavageproducts arise from the action of the protease DPP IV in plasma. Incertain embodiments, GLP-1/glucagon agonist peptides as disclosed areremain stable in plasma at levels up to 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% after 24 hours in plasma at 37° C.

Provided herein is a GLP-1/glucagon agonist peptide comprising the aminoacid sequence:

-   HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30;    wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K    or Y, X15 is D or E. X16 is S or G, X17 is E, R, Q, or K, X18 is R,    S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A    or Q, X27 is E or V, X28 is A or K, and X30 is G or R. (SEQ ID    NO:4). In certain embodiments the isolated peptide shown above is    provided, where X2 is S, X10 is Y or K, X12 is K, E, R, or S, X13 is    K or Y, X15 is D, X16 is S, X17 is E, R, Q, or K, X18 is R, S, or A,    X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and    X30 is G (SEQ ID NO:5). In certain embodiments the isolated peptide    shown above is provided, where X2 is S, X10 is Y or K, X12 is K, E,    R, or S, X13 is K or Y, X15 is D, X16 is S, if X17 is E and X18 is    R, or if X17 is R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is    A, X27 is E or V, X28 is A, and X30 is G (SEQ ID NO: 6 and SEQ ID    NO. 7, respectively). In certain embodiments the isolated peptide    shown above is provided, where X2 is S, X10 is Y, X12 is K, X13 is    K, X15 is D, X16 is S, if X17 is E and X18 is R, or if X17 is R and    X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is    A, and X30 is G (SEQ ID NO: 8 and SEQ ID NO: 9, respectively). In    certain embodiments the isolated peptide shown above is provided,    where X2 is S, X10 is K, X12 is K, E, R, or S, X13 is Y, X15 is D,    X16 is S, if X17 is E and X18 is R, and if X17 is R and X18 is S,    X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30    is G (SEQ ID NO: 10 and SEQ ID NO: 11, respectively). In certain    embodiments the isolated peptide shown above is provided, where X2    is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, if X17 is E    and X18 is R, or if X17 is R and X18 is S, X20 is R, X21 is D, X23    is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 12 and    SEQ ID NO: 13, respectively). In certain embodiments the isolated    peptide shown above is provided, where X2 is S, X10 is K, X12 is R,    X13 is Y, X15 is D, X16 is S, if X17 is E and X18 is R, or if X17 is    R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E,    X28 is A, and X30 is G (SEQ ID NO: 14 and SEQ ID NO: 15,    respectively).

GLP-1/glucagon agonist peptides provided herein include, but are notlimited to G730 (SEQ ID NO: 16), G797 (SEQ ID NO: 17), G849 (SEQ ID NO:18), G933 (SEQ ID NO: 19), G865 (SEQ ID NO: 20), G796 (SEQ ID NO: 21),G812 (SEQ ID NO: 22) and G380 (SEQ ID NO: 23). These GLP-1/glucagonagonist peptides are listed in Table 1:

TABLE 1 GLP-1/Glucagon Peptide Sequences SEQ ID Peptide Sequence NO:G730 HSQGT FTSDY SKXLD SERAR DFVAW LVAGG-amide X13 = K(gE-palm) 16 G797HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-amide X10 = K(gE-palm) 17 G849HSQGT FTSDX SRYLD SRSAR DFVAW LEAGG-amide X10 = K(gE-palm) 18 G933HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-acid X10 = K(gE-palm) 19 G865HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-amide X10 = K(gE-palm) 20 G796HSQGT FTSDX SSYLD SRRAR DFVAW LEAGG-amide X10 = K(gE-palm)  21 G812HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-amide X10 = K(gE-palm) 22 G380HGQGT FTSDY SKYLD SXRAQ DFVQW LVAGG-amide X17 = K(gE-palm) 23 G931HSQGT FTSDY SKXLD SERAR DFVAW LVAGG-acid X13 = K(gE-palm) 24 G934HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-acid X10 = K(gE-palm) 25 G973HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-acid X10 = K(gE-palm) 26 GLP1HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR SEQ ID NO: 2 (7-36 amide)/SEQID NO: 3 (7-37 acid) Glucagon HSQGT FTSDY SKYLD SRRAQ DFVQW LMNT SEQ IDNO: 1 K(gE-Palm) = Lysine with a palmitoyl group conjugated to theepsilon nitrogen, through a gamma glutamic acid linker.

The peptides G797 and G933 both have a glutamate residue at position 12,and maintain robust activity at both the glucagon and GLP-1 receptors,as shown in Example 2. The corresponding residue is lysine in exendin-4and glucagon and is serine in GLP-1. Although this residue is notthought to contact the receptor, changes in charge from positive tonegative may modify the adjacent environment. Furthermore, G797, G849and G933 have a glutamate residue at position 27. Residue 27 is Lysinein exendin 4 and is an uncharged hydrophobic residue in GLP1 (valine)and glucagon (methionine). The lysine of exenatide makes electrostaticinteractions with the GLP1 receptor at residues Glu127 and Glu24 (C. R.Underwood et al J Biol Chem 285 723-730 (2010); S. Runge et al J BiolChem 283 11340-11347 (2008)). While a loss of GLP1R potency might beexpected when the charge at position 27 is changed to negative, thechange is compatible with GLP1R activity in G797, G849, and G933.

Methods of Making.

This disclosure provides a method of making a GLP-1/glucagon agonistpeptide. GLP-1/glucagon agonist peptides provided herein can be made byany suitable method. For example, in certain embodiments theGLP-1/glucagon agonist peptides provided herein are chemicallysynthesized by methods well known to those of ordinary skill in the art,e.g., by solid phase synthesis as described by Merrifield (1963, J. Am.Chem. Soc. 85:2149-2154). Solid phase peptide synthesis can beaccomplished, e.g., by using automated synthesizers, using standardreagents, e.g., as explained in Example 1.

Alternatively, GLP-1/glucagon agonist peptides provided herein can beproduced recombinantly using a convenient vector/host cell combinationas would be well known to the person of ordinary skill in the art. Avariety of methods are available for recombinantly producingGLP-1/glucagon agonist peptides. Generally, a polynucleotide sequenceencoding the GLP-1/glucagon agonist peptide is inserted into anappropriate expression vehicle, e.g., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. The nucleic acid encoding the GLP-1/glucagon agonistpeptide is inserted into the vector in proper reading frame. Theexpression vector is then transfected into a suitable host cell whichwill express the GLP-1/glucagon agonist peptide. Suitable host cellsinclude without limitation bacteria, yeast, or mammalian cells. Avariety of commercially-available host-expression vector systems can beutilized to express the GLP-1/glucagon agonist peptides describedherein.

Modifications, Conjugates, Fusions, and Derivations.

In certain embodiments, GLP-1/glucagon agonist peptides provided hereinare stabilized via amino acid modifications. In certain embodiments, thecarboxyl group of the C-terminal amino acid is amidated. In certainembodiments, the C-terminal amino acid is amidated glycine, e.g., G730,G797, G849, G865, G796, G812, and G380. In certain embodiments, e.g.,G933, the C-terminal glycine is the unmodified acid. In certainembodiments, GLP-1/glucagon agonist peptides are provided in which oneor more amino acid residues are acylated. For example, in certainembodiments GLP-1/glucagon agonist peptides provided herein contain oneor more lysine residues, in which a palmitoyl moiety is attached to theN(epsilon) group. In certain embodiments a linker is incorporatedbetween lysine and the palmitoyl group. This linker can be a gammaglutamic acid group, or an alternative linker such as, but not limitedto, beta alanine and aminohexanoic acid. Different acylation methods maybe used such as addition of cholesterol or myristoyl groups. In certainembodiments, the palmitoyl moiety is added at position 13 (e.g., G730).In certain embodiments, the palmitoyl moiety is added at position 10(e.g., G797, G849, G933, G865, G796, and G812). In certain embodiments,the palmitoyl moiety is added at position 17 (e.g., G380).

The GLP-1/glucagon agonist peptides provided herein, e.g., G730, G797,G849 and G933 can be palmitoylated to extend their half-life byassociation with serum albumin, thus reducing their propensity for renalclearance, as described in Example 1.

Alternatively or in addition, a GLP-1/glucagon agonist peptide asdisclosed herein can be associated with a heterologous moiety, e.g., toextend half-life. The heterologous moiety can be a protein, a peptide, aprotein domain, a linker, an organic polymer, an inorganic polymer, apolyethylene glycol (PEG), biotin, an albumin, a human serum albumin(HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody,an antibody fragment, a single chain antibody, a domain antibody, analbumin binding domain, an enzyme, a ligand, a receptor, a bindingpeptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptidepolymer, a cytokine, and a combination of two or more of such moieties.

For example, GLP-1/glucagon agonist peptides can be fused with aheterologous polypeptide. The peptides can be fused to proteins, eitherthrough recombinant gene fusion and expression or by chemicalconjugation. Proteins that are suitable as partners for fusion include,without limitation, human serum albumin, antibodies and antibodyfragments including fusion to the Fc portion of the antibodies. GLP-1has been fused to these proteins with retention of potency (L. Baggio etal, Diabetes 53 2492-2500 (2004); P. Barrington et al Diabetes, Obesityand Metabolism 13 426-433 (2011); P. Paulik et al American DiabetesAssociation 2012, Poster 1946). Extended recombinant peptide sequenceshave also been described to give the peptide high molecular mass (V.Schellenberger et al Nature Biotechnol 27 1186-1190 (2009); PASylation(EP2173890)). In certain embodiments GLP-1/glucagon agonist peptides areincorporated as the N-terminal part of a fusion protein, with the fusionpartner, e.g., the albumin or Fc portion, at the C-terminal end.GLP-1/glucagon agonist peptides as described herein can also be fused topeptides or protein domains, such as ‘Albudabs’ that have affinity forhuman serum albumin (M. S. Dennis et al J Biol Chem 277 35035-35043(2002); A. Walker et al Protein Eng Design Selection 23 271-278 (2010)).Methods for fusing a GLP-1/glucagon agonist peptides as disclosed hereinwith a heterologous polypeptide, e.g., albumin or an Fc region, are wellknown to those of ordinary skill in the art.

Other heterologous moieties can be conjugated to GLP-1/glucagon agonistpeptides to further stabilize or increase half-life. For chemicalfusion, certain embodiments feature maintenance of a free N-terminus,but alternative points for derivatization can be made. A furtheralternative method is to derivatize the peptide with a large chemicalmoiety such as high molecular weight polyethylene glycol (PEG). A“pegylated GLP-1/glucagon agonist peptide” has a PEG chain covalentlybound thereto. Derivatization of GLP-1/glucagon agonist peptides, e.g.,pegylation, can be done at the lysine that is palmitoylated, oralternatively at a residue such as cysteine, that is substituted orincorporated by extension to allow derivatization. GLP-1/glucagonagonist peptide formats above can be characterized in vitro and/or invivo for relative potency and the balance between GLP-1 and glucagonreceptor activation.

The general term “polyethylene glycol chain” or “PEG chain”, refers tomixtures of condensation polymers of ethylene oxide and water, in abranched or straight chain, represented by the general formulaH(OCH₂CH₂)_(n)OH, where n is an integer of 3, 4, 5, 6, 7, 8, 9, or more.PEG chains include polymers of ethylene glycol with an average totalmolecular weight selected from the range of about 500 to about 40,000Daltons. The average molecular weight of a PEG chain is indicated by anumber, e.g., PEG-5,000 refers to polyethylene glycol chain having atotal molecular weight average of about 5,000.

PEGylation can be carried out by any of the PEGylation reactions knownin the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992 andEuropean patent applications EP 0 154 316 and EP 0 401 384. PEGylationmay be carried out using an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer).

Methods for preparing a PEGylated GLP-1/glucagon agonist peptidesgenerally include the steps of (a) reacting a GLP-1/glucagon agonistpeptide or with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby the moleculebecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s).

Pharmaceutical Compositions

Further provided are compositions, e.g., pharmaceutical compositions,that contain an effective amount of a GLP-1/glucagon agonist peptide asprovided herein, formulated for the treatment of metabolic diseases,e.g., obesity.

Compositions of the disclosure can be formulated according to knownmethods. Suitable preparation methods are described, for example, inRemington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed.,Mack Publishing Co., Easton, Pa. (1995), which is incorporated herein byreference in its entirety. Composition can be in a variety of forms,including, but not limited to an aqueous solution, an emulsion, a gel, asuspension, lyophilized form, or any other form known in the art. Inaddition, the composition can contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Once formulated, compositions of the invention can beadministered directly to the subject.

Carriers that can be used with compositions of the invention are wellknown in the art, and include, without limitation, e.g., thyroglobulin,albumins such as human serum albumin, tetanus toxoid, and polyaminoacids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitisB virus core protein, and the like. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronicacid and the like. Compositions can be sterilized by conventional, wellknown sterilization techniques, or can be sterile filtered. A resultingcomposition can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. Compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamineoleate, etc.

Method of Treating Obesity, Model Systems

GLP-1/glucagon agonist peptides can combine the effect of glucagon e.g.,inhibition of food intake or regulation of glucose levels with theeffect of GLP-1 e.g., inhibition of gastric motility, or promotion ofinsulin release. They can therefore act to accelerate elimination ofexcessive adipose tissue, induce sustainable weight loss, and improveglycemic control. GLP-1/glucagon agonist peptides can also act to reducecardiovascular risk factors such as high cholesterol, and highLDL-cholesterol or abnormal HDL/LDL ratios.

This disclosure provides a method of treating obesity or anobesity-related disease or disorder, comprising administering to asubject in need of treatment a GLP-1/glucagon agonist peptide asdisclosed herein. Further provided is a GLP-1/glucagon agonist peptidefor treatment of obesity or an obesity-related disease or disorder.Further provided is use of a GLP-1/glucagon agonist peptide as providedherein in the manufacture of a medicament for the treatment of obesityor an obesity-related disease or disorder.

GLP-1/glucagon agonist peptides provided herein can be administered forpreventing weight gain, promoting weight loss, reducing excess bodyweight or treating obesity (e.g. by control of appetite, feeding, foodintake, calorie intake, and/or energy expenditure), including morbidobesity. In addition, GLP-1/glucagon agonist peptides provided hereincan be used for treatment of other obesity-related metabolic disorders.Examples of other obesity-related disorders include without limitation:insulin resistance, glucose intolerance, pre-diabetes, increased fastingglucose, type 2 diabetes, hypertension, dyslipidemia (or a combinationof these metabolic risk factors), glucagonomas, cardiovascular diseasessuch as congestive heart failure, atherosclerois, arteriosclerosis,coronary heart disease, or peripheral artery disease, stroke,respiratory dysfunction, or renal disease.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. As provided herein, beneficial or desired clinical results fromthe disclosed GLP-1/glucagon agonist peptides include, withoutlimitation, reduced body weight, decreased weight-gain, reducedappetite, reduced or stabilized serum glucose and serum insulin levels,amelioration, palliation, stabilization, diminishment of extent ofobesity-related diseases, or a delay or slowing of obesity-relateddisease progression. “Treatment” refers to both therapeutic treatmentand prophylactic or preventative measures in certain embodiments. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. By treatment is meantinhibiting or reducing an increase in obesity-related symptoms (e.g.weight gain) when compared to the absence of treatment, and is notnecessarily meant to imply complete cessation of the relevant condition.

The route of administration of GLP-1/glucagon agonist peptides providedherein can be, for example, oral, parenteral, by inhalation or topical.The term parenteral as used herein includes, e.g., intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, orvaginal administration. Another example of a form for administration isa solution for injection, in particular for intravenous or intraarterialinjection or drip. GLP-1/glucagon agonist peptides provided herein canbe administered as a single dose or as multiple doses. In certainembodiments, a GLP-1/glucagon agonist peptide is administered bysubcutaneous injection.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis. Dosage regimens also can be adjustedto provide the optimum desired response (e.g., a therapeutic orprophylactic response).

The amount of a GLP-1/glucagon agonist peptide to be administered can bereadily determined by one of ordinary skill in the art without undueexperimentation given the disclosure herein. Factors influencing themode of administration and the respective amount of a GLP-1/glucagonagonist peptide include, but are not limited to, the severity of thedisease (e.g., the extent of obesity), the subject's history, and theage, height, weight, health, and physical condition of the subjectundergoing therapy. Similarly, the amount of a GLP-1/glucagon agonistpeptide to be administered will be dependent upon the mode ofadministration and whether the subject will undergo a single dose ormultiple doses of this agent. In certain embodiments, GLP-1/glucagonagonist peptides provided herein can be administered once per day viainjection.

Kits

In yet other embodiments, the present disclosure provides kitscomprising GLP-1/glucagon agonist peptides, that can be used to performthe methods described herein. In certain embodiments, a kit comprises aGLP-1/glucagon agonist peptide disclosed herein in one or morecontainers. One skilled in the art will readily recognize that thedisclosed GLP-1/glucagon agonist peptides can be readily incorporatedinto one of the established kit formats which are well known in the art.

EXAMPLES Example 1: Synthesis, Modifications, and Characterization ofGLP-1/Glucagon Agonist Peptides List of Abbreviations

-   -   Boc: tert-butyloxycarbonyl    -   tert-Bu; tert-butyl    -   DCM: dichloromethane    -   DIC: diisopropylcarbodiimide    -   Fmoc: 9-fluorenylmethoxycarbonyl    -   HOBt: 1-hydroxybenzotriazole    -   HPLC: High Performance Liquid Chromatography    -   Mtt: 4-methyltrityl    -   NMP: N-methylpyrrolidone    -   Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl    -   TFA: trifluoroacetic acid    -   TIS: triisopropylsilane    -   Trt: triphenylmethyl, trityl

GLP-1/glucagon agonist peptides were synthesized as follows. Elongationof peptide chains on NovaSyn TGR or preloaded Fmoc-Wang resin(NovaBiochem) was performed with a PRELUDE™ solid phase peptidesynthesizer (Protein Technologies, Tucson, Ariz., USA).Manufacturer-supplied protocols were applied for coupling of thehydroxybenzotriazole esters of amino acids in N-methylpyrolidone (NMP).The fluorenylmethoxycarbonyl (Fmoc) group was used for the semipermanentprotection of alpha-amino groups of amino acids, whereas the side chainswere protected with tert-butyl (tert-Bu) for serine, threonine, asparticacid, glutamic acid, tyrosine, and2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,and trityl (Trt) for histidine. The N-terminal amino group of histidinein position 1 was protected with tert-butyloxycarbonyl group (Boc).Lys(Mtt) was incorporated into the peptide chain when a subsequentchemical modification of the side chain was required.

Upon completion of the peptide chain elongation, the Mtt group wasremoved by washing the peptide-resin with DCM containing 2% TFA and 5%TIS (10×7 ml, each 0.5 min) Coupling of a lipid moiety to the side chainof Lys was performed on the PRELUDE™ peptide synthesizer using DIC as acoupling reagent in the presence of HOBt.

Peptides were cleaved from the resin using mixture of TFA:TIS:water(95:2.5:2.5). After 2 h at room temperature, the peptidyl resin wasfiltered, washed with TFA and combined filtrates were evaporated todryness in vacuo. The residue was triturated with ether, and theprecipitate which formed was filtered, washed with ether, and dried. Thecrude peptides were dissolved in 5% acetic acid in water and analyzed byreverse-phase high-pressure liquid chromatography on a Polaris 3 C8-Acolumn attached to Varian 920-LC system. A standard gradient system of10 to 90% buffer B over the course of 15 min was used for analysis.Buffer A was 0.1% TFA in water and buffer B was 0.1% TFA inacetonitrile. HPLC profiles were recorded at 210 nm. Preparativeseparations were performed on Varian ProStar system with asemipreparative C18 RP XBridge Waters column. The above-describedsolvent system of water and acetonitrile, in a gradient of 30 to 70%buffer B over the course of 30 min, was used for separation. Thechromatographically homogenous products (>97% pure) were analyzed byelectrospray mass spectrometry (MassLynx, Waters).

Example 2: In Vitro Studies Glucagon and GLP-1 Receptor Mediated cAMPProduction

Biological Activity of Peptides in Cell-Based cAMP Activity Assay (Assay1):

The biological activity of GLP-1/glucagon agonist peptides synthesizedby the method of Example 1 were tested for biological activity, e.g.,stimulation of one or more cellular receptor responses, by the followingmethods. Stable cell lines expressing human, mouse, rat, or dog GLP-1receptor (GLP-1R), glucagon receptor (GCGR) or glucose-dependentinsulinotropic peptide (gastric inhibitory polypeptide) receptor (GIPR)were generated in HEK293s or CHO cells by standard methods. Peptideactivation of these various receptors results in downstream productionof cAMP second messenger which can be measured in a functional activityassay.

-   -   cAMP assays were performed using “assay medium”:    -   Assay Medium: 10% FBS in DMEM (Gibco #41966), containing 0.5 mM        IBMX (Sigma #I7018).        Low protein binding 384-well plates (Greiner #781280) were used        to perform eleven 1 in 5 serial dilutions of test samples which        were made in assay medium. All sample dilutions were made in        duplicate.

A frozen cryo-vial of cells expressing the receptor of interest wasthawed rapidly in a water-bath, transferred to pre-warmed assay mediaand spun at 240×g for 5 minutes. Cells were re-suspended in assay mediaat an optimised concentration (e.g. hGCGR cells at 1×10⁵ cells/ml,hGLP-1R and hGIPR cells at 0.5×10⁵ cells/ml).

From the dilution plate, a 5 μL replica was stamped onto a blackshallow-well u-bottom 384-well plate (Corning #3676). To this, 5 μL cellsuspension was added and the plates incubated at room temperature for 30minutes.

cAMP levels were measured using a commercially available cAMP dynamic 2HTRF kit (Cisbio, Cat #62AM4PEJ), following the two step protocol as permanufacturer's recommendations. In brief; anti-cAMP cryptate (donorfluorophore) and cAMP-d2 (acceptor fluorophore) were made up separatelyby diluting each 1/20 in conjugate & lysis buffer provided in the kit. 5μL anti-cAMP cryptate was added to all wells of the assay plate, and 5μL cAMP-d2 added to all wells except non-specific binding (NSB) wells,to which conjugate and lysis buffer was added. Plates were incubated atroom temperature for one hour and then read on an Envision (PerkinElmer) using excitation wavelength of 320 nm and emission wavelengths of620 nm & 665 nm.

Sequences of synthesized GLP-1/glucagon agonist peptides and their EC50values determined in cAMP assays, performed in “assay medium,” are shownin Table 2. All peptides in Table 2 were synthesized with a C-terminalamide. Additional GLP-1/glucagon agonist peptides were synthesized witha C-terminal acid and EC50 values determined in cAMP assays, performedin “assay medium,” are shown in Table 3. EC50 for additionalGLP-1/glucagon agonist peptides, performed in “assay medium,” are shownin Table 4. All peptides in Table 4 have a C-terminal amide, unless theyare denoted as ‘acid’ in which case they have a C-terminal acid.

TABLE 2 cAMP activity of GLP-1/glucagon agonist peptides with C-terminalamide (assay 1) Assay in Assay Medium Human Human Human GlucR EC50 GLP1REC50 GIPr EC50 Peptide M M M G730 6.23E−12 1.8E−11 4.5E−08 G797 6.14E−121.4E−11 3.4E−09 G849 2.26E−12 9.0E−12 1.7E−08 G865 1.26E−11 8.3E−122.2E−08 G796 1.76E−12 1.3E−11 1.4E−08 G812 8.17E−12 1.1E−11 2.7E−09 G3802.17E−10 7.7E−11 1.3E−07 GLP1 8.1E−11 Glucagon  3.3E−12

TABLE 3 cAMP activity of GLP-1/glucagon agonist peptides with C-terminalacid (assay 1) Human GlucR Human GLP1R Human GIPr EC50 EC50 EC50 PeptideM M M G931 1.78E−11 1.30E−10 0.00E+00 G933 5.92E−12 3.20E−11 9.70E−09G934 6.30E−12 1.80E−11 3.60E−09 G973 8.90E−12 1.20E−11 4.70E−08

TABLE 4 cAMP activity of additional GLP-1/glucagon agonist peptides(assay 1) Site and nature of palmitoylation, hGlucR hGLP1R hGIPRSubstitutions into EC50 M EC50 M EC50 M parent sequence Parent sequenceHSQGT FTSDY SKYLD SRRAQ DFVQW LVAGG Peptides in this section all containLVAGG at residues 26 to 30 Glucagon  3.3E−12  4.2E−09 1.99E−07 GLP11.53E−07  8.1E−11 1.53E−07 g715 2.53E−12 2.04E−11 9.98E−10 K(gE-palm)10g716 2.46E−09 1.29E−08 1.18E−08 K(gEpalm)11 g702 1.49E−09 3.35E−090.00E+00 K(gEpalm)12, E17 g728 2.44E−09 1.69E−10 3.95E−07 K(gEpalm)12,E17 R20 A24 g729 3.19E−11 7.29E−11 2.09E−07 K(gEpalm)13 E17 g7301.50E−11 3.95E−11 5.66E−08 K(gEpalm)13 E17 R20 A24 g875 1.29E−102.98E−11 2.90E−08 K(gEpalm)13 R20 A24, E17 Aib2 g841 no data K(gEpalm)13R20 A24, S18 R12 acid g802 1.81E−09 9.64E−11 9.12E−08 K(gEpalm)13, R20A24, E17, E12 g820 1.17E−11 3.39E−11 7.11E−08 K(gEpalm)13, R20 A24, E17,R12 g842 8.31E−12 5.12E−11 8.83E−08 K(gEpalm)13, R20 A24, E17, R12 acidg733 6.20E−08 2.31E−11 8.17E−07 K(gEpalm)14, G2 E3 g803 1.08E−112.96E−11 3.29E−08 K(gEpalm)14, R20 E24, S18 g843 no data K(gEpalm)14,R20 E24, S18 R12 acid g732 3.96E−11 2.32E−11 2.94E−08 K(gEpalm)14, R20A24, E17 G2 g777 1.24E−12 2.74E−11 4.53E−09 K(gEpalm)14, R20 A24, E17g844 no data K(gEpalm)14, R20 A24, E17 R12 Aib2 acid g845 no dataK(gEpalm)14, R20 A24, E17 R12 acid g821 4.63E−12 5.58E−11 1.40E−08K(gEpalm)14, R20 A24, E17, R12 g846 3.41E−11 4.38E−11 1.18E−08K(gEpalm)14, R20 A24, E17, E12 g731 2.77E−11 4.22E−11 4.07E−08K(gEpalm)14, E12 g670 8.00E−12 2.03E−11 1.49E−08 K(gEpalm)14, S18 g3351.05E−11 7.33E−11 5.82E−07 K(gE-palm)17 g336 1.77E−12 3.66E−11 1.96E−08K(gE-gE-palm)17 g384 4.29E−11 2.72E−11 1.70E−08 K(gEpalm)17, Aib2 g3803.62E−10 1.00E−10 6.09E−07 K(gEpalm)17, G2 g736 9.19E−10 8.54E−110.00E+00 K(gEpalm)17, G2, A20 A24 g381 1.93E−09 9.08E−11 5.45E−07K(gEpalm)17, E3 g678 4.52E−09 1.06E−10 1.23E−07 K(gEpalm)17, G2 E20g599, g688 6.98E−11 1.20E−10 1.12E−07 K(gEpalm)17, E20 g679 1.89E−101.35E−10 1.17E−07 K(gEpalm)17, G2 E24 g600, g689 5.47E−12 6.66E−118.28E−08 K(gEpalm)17, E24 g680 3.68E−09 1.95E−10 9.67E−08 K(gEpalm)17,G2 E20 E24 g639 8.21E−08 2.44E−10 8.21E−08 K(gEpalm)17, S2 E3 E20 E24g681 3.99E−08 2.83E−10 1.24E−07 K(gEpalm)17, G2, E3 E20 E24 g7203.52E−10 5.34E−11 0.00E+00 K(gEpalm)17, G2 R20 E24 R12 g660 1.52E−091.06E−09 3.32E−07 K(gEpalm)17, G2 R20 E24 g835 4.24E−10 1.91E−109.72E−08 K(gEpalm)17, R20 E24, E12 g776 4.65E−12 7.02E−11 4.79E−08K(gEpalm)17, R20 E24 g823 9.48E−12 9.73E−11 8.42E−08 K(gEpalm)17, R20E24, R12 g867 7.04E−12 4.48E−11 4.17E−08 K(gEpalm)17, R20 A24 g7369.20E−10 8.54E−11 0.00E+00 K(gEpalm)17, A20 A24, G2 g737 7.34E−078.14E−11 0.00E+00 K(gEpalm)17, A20 A24, G2 E3 g675 3.84E−08 1.51E−101.61E−06 K(gEpalm)17, E12 R20 A24 G2 Parent sequence HSQGT FTSDY SKYLDSRRAQ DFVQW LEAGG Peptides in this section all have the sequence LEAGGfrom residue 26 onwards unless otherwise stated, e.g. LERGG g7174.55E−13 5.77E−12 1.48E−09 K(gEpalm)10, LEAGG g796 1.81E−12 1.40E−111.74E−08 K(gEpalm)10, LEAGG, R20 A24 S12 g847 no data K(gEpalm)10,LEAGG, R20 A24 S18 E12 Aib2 acid g797 9.64E−12 2.26E−11 4.64E−09K(gEpalm)10, LEAGG, R20 A24 E17 E12 g798 5.10E−13 9.07E−12 1.51E−09K(gEpalm)10, LEAGG, R20 A24 E17 g848 9.66E−13 9.42E−12 2.77E−09K(gEpalm)10, LEAGG, R20 A24 E17 R12 g849 2.28E−12 9.07E−12 1.81E−08K(gEpalm)10, LEAGG, R20 A24 S18 R12 g701 3.83E−09 7.40E−09 0.00E+00K(gEpalm)12, LERGG, G2 E17 g840 5.30E−12 1.45E−10 1.02E−07 LEAGG, R20A24, E17 g824 1.05E−12 4.71E−11 5.74E−08 K(gEpalm)14, LEAGG, R20, E24g780 7.92E−13 1.20E−11 6.40E−08 K(gEpalm)14, LEAGG, R20 A24 g6014.93E−13 3.98E−11 7.41E−08 K(gEpalm)14, LEAGG g816 1.10E−12 3.16E−112.00E−08 K(gEpalm)14, LEAGG, E17 g817 1.68E−12 2.51E−11 1.52E−08K(gEpalm)14, LEAGG, A18 g876 1.04E−11 8.63E−11 7.90E−08 K(gEpalm)14,LEAGG, R20, E24, E12 g805 1.44E−12 2.28E−11 9.97E−08 K(gEpalm)14, LEAGG,R20 E24 g850 2.19E−12 2.12E−11 8.96E−08 K(gEpalm)14, LEA, R20, A24, S18R12 g836 1.55E−11 1.24E−10 1.00E−07 K(gEpalm)14, LEAGG, R20 E24, E17g804 1.95E−12 7.15E−11 9.97E−08 K(gEpalm)14, LEA, R20, A24 g618 no dataK(Ahx-palm)20, LEKGR g781 2.86E−12 1.04E−10 4.02E−07 K(gEpalm)16, LEAGG,R20 A24 g782 1.56E−10 2.54E−11 1.43E−06 K(gEpalm)18, LEAGG, R20 A24 g7443.92E−11 2.45E−09 0.00E+00 K(gE-palm)20, LEAGG g746 3.54E−11 1.15E−080.00E+00 K(gE-palm)24, LEAGG g747 9.42E−11 3.16E−09 1.04E−06K(gE-palm)31, LEAGG g512 6.06E−11 9.80E−11 4.07E−07 K(gEpalm)17, LEAGG,G2, g513 7.23E−10 1.75E−10 2.98E−07 K(gEpalm)17, LEAGG, E3, g7348.28E−08 6.95E−11 1.17E−06 K(bA-palm)17, LEAGG, R20 A24, E3 E12 g8372.13E−10 4.67E−10 1.14E−07 K(gE-palm)17, LEAGG, R20 A24 E12 g8385.68E−12 2.37E−11 8.43E−08 K(Ahx-palm)17, LEAGG, R20 A24 E12 g7839.11E−11 4.24E−11 8.46E−07 K(bA-palm)17, LEAGG, R20 A24 E12 g851 no dataK(bA-palm)17, LEAGG, R20 A24, R12 acid g852 no data K(bA-palm)17, LEAGG,R20 A24, R12 Aib2 acid g819 2.34E−12 1.80E−11 1.03E−07 K(bA-palm)17,LEAGG, R20 A24 g536 4.78E−12 7.45E−11 0.00E+00 g600 5.47E−12 6.66E−111.24E−07 K(gE-palm)17, LVAGG, E24 g599 9.62E−11 8.76E−11 1.13E−07K(gE-palm)17, LVAGG, E20 Parent sequence HSQGT5 FTSDY10 SKYLD15 SRRAQ20DFVQW25 LERGG-amide Peptides in this section all have the sequence LERGGfrom residue 26 onwards unless otherwise stated, e.g. LENT g825 3.67E−121.91E−11 8.67E−08 K(Ahx-palm)17, LENT, R20 E24, E12 g588 7.23E−111.10E−10 9.80E−08 K(gEpalm)17, LERGG, G2, g614 3.65E−12 9.31E−129.93E−08 K(Ahx-palm)17, LERGG, E12 g684 1.64E−10 1.51E−11 1.46E−07K(Ahx-palm)17, LERGG, R20 A24 E12 G2 g721 3.23E−09 4.11E−10 9.79E−07K(gE-palm)17, LERGG, R20 A24 E12 G2 g724 3.09E−08 1.90E−11 9.33E−07K(Ahx-palm)17, LERGG, R20 A24 E12 G2 E3 g772 1.84E−10 2.92E−10 1.54E−06K(gE-palm)17, LERGG, R20 A24 E12 g795 1.10E−10 7.34E−11 5.79E−07K(bA-palm)17, LERGG, R20 A24 E12 g794 4.69E−12 1.57E−11 3.22E−08K(Ahx-palm)17, LERGG, R20 A24 E12 g826 4.23E−12 2.93E−11 5.80E−08K(Ahx-palm)17, LERGG, R20 A24 E12 acid g727 2.18E−10 2.63E−11 1.77E−07K(Ahx-palm)17, LERGG, R20 A24 E12 G2 acid g683 3.72E−10 1.59E−111.26E−07 K(Ahx-palm)17, LERGG, A20 A24 E12 G2 g722 1.11E−08 4.26E−101.67E−06 K(gE-palm)17, LERGG, A20 A24 E12 G2 g725 5.99E−08 2.52E−111.48E−06 K(Ahx-palm)17, LERGG, A20 A24 E12 G2 E3 g818 8.90E−12 2.10E−119.40E−08 K(Ahx-palm)17, LERGG, A20 A24 E12 g682 1.95E−10 1.43E−111.22E−07 K(Ahx-palm)17, LERGG, R20 E24 E12 G2 g723 8.95E−09 3.30E−107.61E−07 K(gE-palm)17, LERGG, R20 E24 E12 G2 g726 1.31E−08 7.91E−122.15E−07 K(Ahx-palm)17, LERGG, R20 E24 E12 G2 E3 g771 5.51E−12 1.75E−113.71E−08 K(Ahx-palm)17, LERGG, R20 E24 E12 g617 no data K(Ahx-palm)20,LERGG, G2, E12, g787 4.36E−11 6.65E−09 0.00E+00 K(Ahx-palm)20, LERGG,A24 E17 g806 9.9E−12 1.71E−10 1.05E−07 K(Ahx-palm)21, LERGG, A18 g616 nodata K(Ahx-palm)24, LERGG, G2, E12 g701 3.83E−09 7.4E−09 0.00E+00K(gEpalm)12, LERGG, G2 E17 Parent sequence HSQGT5 FTSDY10 SKYLD15SRRAQ20 DFVQW25 LVAGG extension Peptides in this section have theresidues noted C-terminal to residue 30 and a C-terminal amide hGlucRhGLP1R hGIPR EC50 M EC50 M EC50 M Extension to sequence g316 1.06E−113.14E−11 3.65E−09 SSGGSS g317 0.00E+00 2.63E−09 0.00E+00 SSGGSS K g3189.04E−09 1.14E−09 0.00E+00 SSGGSSK(palm) g402 5.96E−11 8.57E−11 0.00E+00SGSGSG g319 1.04E−11 3.61E−11 0.00E+00 PSSGA PPPSK g320 3.20E−129.38E−12 1.01E−09 PSSGA PPPSK(palm) g315 5.04E−12 2.73E−11 1.97E−08 GGGGg325 1.03E−11 2.61E−11 0.00E+00 GGGGK g326 2.82E−12 2.47E−11 1.26E−08GGGGK(palm) g327 2.32E−12 1.93E−11 1.28E−08 GGGGK(gEpalm) g321 2.79E−112.72E−11 6.41E−09 KNNRNNIAK g322 3.55E−12 1.06E−11 1.72E−09KNNRNNIAK(palm) Abbreviations: K(gE-palm) = Lysine with a palmitoylgroup conjugated to the epsilon nitrogen, through a gamma glutamic acidlinker; K (Ahx-palm) = Lysine with a palmitoyl group conjugated to theepsilon nitrogen, through an aminohexanoic acid linker; K(bA-palm) =Lysine with a palmitoyl group conjugated to the epsilon nitrogen,through a beta alanine acid linker; Aib, aminoisobutyric acid. K(palm) =Lysine with a palmitoyl group directly conjugated to the epsilonnitrogen.

Glucagon and GLP-1 Receptor Mediated cAMP Production Assays in Presenceof Plasma Concentrations of Serum Albumin (Assay 2).

Agonist potency determinations for peptides inducing cAMP productionwere measured in CHO cells expressing human, rat or mouse glucagonreceptors (abbreviated to GlucR or GCGR) or GLP-1 receptors in thepresence of human, rat or mouse serum albumin at 4.4, 3.2 and 3.2%respectively, as follows.

CHO cells with stable recombinant expression of the human, mouse or ratGlucR or GLP-1 receptor were cultured in DMEM 10% FBS and geneticin (100μg/ml). Cryopreserved cells stocks were prepared in 1× cell freezingmedium-DMSO serum free (Sigma Aldrich) at 2×10⁷/vial and stored at −80°C. Cells were rapidly thawed at 37° C. and then diluted in to assaybuffer (DMEM) containing serum albumin at 4.4, 3.2 and 3.2% for human,rat, and mouse serum albumin respectively. Peptides were seriallydiluted in DMSO and then diluted 100 fold into DMEM containing serumalbumin at stated final concentration. Diluted peptides were thentransferred into 384 black shallow well microtitre assay plates. Cellswere added to the assay plates and incubated for 30 min at roomtemperature. Following incubation the assay was stopped and cAMP levelsmeasured using the HTRF® dynamic d2 cAMP assay kit available from CisBioBioassays, as per the manufacturers guidelines. Plates were read onPerkin Elmer ENVISION® fluorescence plate readers. Human and rat serumalbumin were purchased from Sigma Aldrich and mouse serum albumin fromEquitech Bio Ltd.

Data was transformed to % Delta F as described in manufacturer'sguidelines and analysed by 4-parameter logistic fit to determine EC₅₀values. Assay 2 EC₅₀ values for selected peptides are shown the Table 5.The assay 2 EC50 values determined are dependent on both the intrinsicpotency of the peptides tested at the GLP1 and glucagon receptors in therecombinant cell lines and on the affinity of the peptide for serumalbumin, which determines the amount of free peptide. Association withserum albumin increases the EC50 value obtained. The fraction of freepeptide at plasma concentrations of albumin and the EC50 at 0% HSA canbe calculated based on the variation in cAMP generation with the HSAconcentration. For instance, G730 and G933 gave values of 0.85% and0.29% for free peptide at 4.4% HSA and 7 pM and 6 pM for the EC50 at theGLP1R at 0% HSA respectively. G797 and G849 give values of 0.82% and0.48% for free peptide at 4.4% HSA and 7 pM and 2 pM for the EC50 at theGLP1R at 0% HSA respectively. To compare the balance of activities atthe GLP1R and GlucR between different peptides and across differentconditions, these can be correlated using the calculation below, wherethe EC50's are related to those of the natural ligands.

TABLE 5 EC50 Potencies for GLP-1/Glucagon Agonist Peptides in thePresence of Plasma Concentrations of Serum Albumin (Assay 2) Assay in4.4% Human Assay in 3.2% Mouse Serum Assay in 3.2% Rat Serum SerumAlbumin Albumin Albumin Human Human Mouse Mouse Rat Rat GLP1R GlucRHuman GLP1R GlucR Mouse GLP1R GlucR Rat EC50 EC50 GlucR/GLP1R EC50 EC50GlucR/GLP1R EC50 EC50 GlucR/GLP1R Peptide pM pM Ratio¹ pM pM Ratio¹ pMpM Ratio¹ G730 455 402 0.122 1100 5460 0.04 81 45080 0.06 G797 739 11370.07 290 764 0.08 60 23170 0.08 G849 172 79 0.235 88 103 0.17 44 40550.33 G933 943 564 0.179 540 377 0.29 136 15500 0.27 G865 150 570 0.02796 1100 0.021 18 87100 0.01 G796 140 53 0.275 130 34 0.78 23 2000 0.36G812 316 764 0.044 130 947 0.032 19 14100 0.04 G380 6543 53590 0.01315000 576000 0.006 GLP1 25 21 1.9 Glucagon 2.7 9700 4.97 557 60¹GlucR/GLP1R ratios were determined as follows: Relative Potency GlucR =EC50 Glucagon/EC50 Tested peptide Relative Potency GLP1R = EC50GLP1/EC50 Tested peptide GlucR/GLP1R Ratio = Relative PotencyGlucR/Relative Potency GLP1R

Stability Testing of Peptides in Plasma.

The stability in plasma of the peptides G730, G797, G849 and G933 wasdetermined as follows.

Stock solutions of the peptides of about 200 μmol/L was prepared byweighing solid peptide into a Eppendorf Low Bind Tube and dissolved inDMSO. 10 μL of stock solutions were added to 990 μL of plasma in anEppendorf Low Bind Tube, resulting in initial concentrations of thepeptides in plasma of about 2 μmol/L. The frozen blank plasma fromhuman, rat and mouse had been thawed and heated to a temperature of 37°C. before addition of the stock solution. The spiked plasma samples weregently mixed and allowed to equilibrate for about 5 minutes before startof experiment. The plasma samples were incubated for 48 hours in aGalaxyR CO₂ incubator at 37° C. Sampling (30 μL) was performed at 0, 1,2, 6.5, 17, 24 and 48 hours. The samples were stored at −70° C. untilanalysis.

Plasma samples were assayed as follows. The 30 μL plasma samples wereprotein precipitated with 180 ml of cold ethanol in a 96-well low bindplate (Eppendorf Protein LoBind). After mixing and centrifugation 100 μlthe supernatant was transferred to a new plate and 1 μl was injectedonto an analytical column.

The analysis was performed using a μLC-system (LC Exigent μLC) coupledto a medium high resolution mass spectrometer (Perkin Elmer PenTOF) withpositive electrospray ionisation. The analytical column was a 5 cm, 1 mmAgilent Poroshell (custom made) C18-column with a particle size of 2.7μm. Flow: 0.1 ml/min using a slow reversed phase gradient. Mobile phasesused were acetonitrile and water containing 0.1% formic acid.

The resulting data were manually evaluated for the following degradationproducts: +1 product (acid) and the DPP IV-cleavage product. Productswith +1 mass may arise from deamidation at amide groups of glutamine orat the C-terminus Cleavage products arise from the action of theprotease DPP IV in plasma. Both the degradation of the peptides andformation of peptide products were reported in percentage of the initialpeptide concentration. Peaks were integrated and % remaining peptide wascalculated: (peak area/peak area 0H)*100. Data for the 24 h time pointis shown in Table 6. Levels of deamidation and DPP IV cleavage were lowfor G797 and G933.

TABLE 6 Peptide Stability in Plasma Plasma Stability in Plasma Stabilityin Human Plasma Stability in Rat Mouse Plasma at 24 h Plasma at 24 hPlasma at 24 h % DPP % DPP % DPP cleaved cleaved cleaved product/+1product/+1 product/+1 % % DPP % % DPP % % DPP stable +1 cleaved stable+1 cleaved stable +1 cleaved Peptide peptide prod product peptide prodproduct peptide prod product G730 65 15 14/5 100 <1 <1 24 58 2 G797 84<1 1 85 <1 <1 60 <1 1 G849 38 <1 22 100 <1 <1 69 16 3 G933 83 1 86 <1 85<1

Solubility

Peptide solubility was assessed in a variety of buffer species within apH range of 4.5 to 8.0, as follows. Dried powder forms of theGLP-1/Glucagon agonist peptides were reconstituted in various buffers atroom temperature. The absorbance was measured at 280 nm using NanoDrop2000 spectrophotometer and the peptide concentration was calculatedusing the following equation:c=(A ₂₈₀ *M _(w))/ε

where:

-   -   c—concentration ε—extinction coefficient Mw—molecular weight        A₂₈₀—Absorbance at 280 nm    -   ε=(1×Trp=5560)+(1×Tyr=1200)

The Results are shown in Table 7. Each of the peptides was soluble at0.8 mg/ml over a range of pH (6.5 to 8.5). G730 was soluble in a pHrange 4.5 to 8.0, G797 was soluble in a pH range of 6. to 8.0, and G933was soluble in a pH range of 6 to 8.0. The solubility of G933 was testedin a number of different buffer systems, also shown in Table 7. G933 wassoluble at 1 mg/ml in at least the following buffer systems: histidine(pH 6 and 7; ionic strength: 0.25 to 100 mM), sodium phosphate (pH6-7.5; ionic strength: 0.25 to 100 mM), and tris/hydroxymethylaminomethane (pH 7-9; ionic strength: 0.25 to 100 mM).

TABLE 7 Peptide solubility profile (Ionic Strength of all buffers: 100mM) Conc. (mg/ml) A280 Target 1 mg/ml Buffer G730 G797 G849 G933Glutamate pH 4.5 0.83  0.023 NA  0.02 Acetate pH 5 NA NA NA  0.03Succinate pH 5 NA NA NA 1.1 Phosphate, pH = 6 0.14 0.84 0.06 1.2Histidine pH 6 NA NA NA 1.2 Phosphate pH 6.5 0.83 0.84 NA NA Phosphate,pH 7.0 NA NA NA 1.1 Histidine, pH 7.0 NA NA NA 1.1 Phosphate pH 7.5 0.850.86 NA 1.2 Tris pH 7.5 0.83 0.89 0.89 1.2 Tris pH 8.0 1.1  0.83 0.891.2

Formulations.

Peptide solubility was assessed in three different isotonicformulations:

-   1. Default Formulation (DF)=0.1M Tris pH 7.5, 150 mM Mannitol. Final    formulation pH=7.2-   2. Back up formulation 1 (BF1)=0.05M Tris, 50 mM Arginine/Proline.    Final formulation pH=8.0-   3. Back up formulation 2 (BF2)=Sodium Phosphate buffer (pH8)/1.85%    W/V propylene glycol. Final formulation pH=7.0

Solubility was measured as detailed above, and the results are shown inTable 8. G730, G797 and g933 were soluble to at least 5 mg/ml in the DF,the maximum solubility of G849 in DF was 3.7 mg/ml, G797 was soluble toat least 10 mg/ml in BF1, and G933 was soluble to at least 10 mg/ml inBF2.

TABLE 8 Peptide Solubility in Formulation 10 mg/ml Lead Formulationsolubility Candidate Concentration Formulation (BF2) G730 5 mg/ml DF noG797 5 mg/ml DF/BF1 yes G849 3.7 mg/ml   DF n/a G933 5 mg/ml DF yesConcentration Determined by A280 nm

The stability of the DF was evaluated by measuring purity reversed phaseultra-performance liquid chromatography (RP UPLC), within one month. Thestorage conditions were 5° C., 25° C., 40° C. and −80° C. The resultsare shown in Tables 9 and 10.

TABLE 9 Peptide formulation purity after 1 month in stability conditionsPeptide 5° C. 25° C. 40° C. minus 80 C. G730_DF 97.7 96.1 86.1 97.7G797_BF1 98.72  98.84  77.54 NA G849_DF 95.5 NA NA NA G933_DF 97.8 95.988.9 98.9

TABLE 10 Peptide formulation purity loss (% compared to T0) after 1month in stability conditions Lead Candidate 5° C. 25° C. 40° C. minus80 C. G730_DF 0.82 2.43 12.54 0.3 G797_BF1 0.24 0.12 21.65 0.3 G849_DFn/a n/a n/a n/a G933_DF 0.3 2.2 9.3 (−)0.8The peptides all showed acceptable properties with respect tosolubility, formulatability and stability

Example 3: In Vivo Studies

G730, G797, and G812 (Study A).

Selected GLP-1/glucagon agonist peptides disclosed herein were tested ina diet induced obesity (DIO) mouse model, as follows. FemaleC57/B16JHsdO1a (obtained from Harlan Laboratories, UK) were started on ahigh fat diet of D12492 (Research Diets, N.J., USA) and a chocolateconfection, delicato ball (Delicata Bakverk, Sweden) at 9-11 weeks ofage, and were maintained on the diet for 16 weeks prior to arrival tothe animal facility, during a three week acclimatizion period and duringdrug treatment, caloric content of the two components of the diet isshown in Table 11. The mice were divided into 9 groups (n=5-6), andtreatment was started at 29 weeks of age. The treatment groups anddosing are shown in Table 12.

TABLE 11 Content of DIO Diet Carbo- Total Protein hydrate Fat Kcal fatKcal/ Product (%) (%) (%) (%) gram Delicatoball 5 53 31 54 5.05(Delicata Bakverk AB, Huddinge, Sweden) D12492 26.2 26.3 34.9 60 5.24(research Diets, NJ, USA)

TABLE 12 Treatment Groups for Study A Peptide Dose # of Animals VehicleNA 6 Liraglutide 26.6 nmol/kg   6 G730 10 nmol/kg 6 G730 20 nmol/kg 5G730 50 nmol/kg 6 G797  5 nmol/kg 5 G797 20 nmol/kg 6 G797 50 nmol/kg 6G812 20 nmol/kg 5

GLP-1/glucagon agonist peptides G730, G797, and G812, as well asLiraglutide were formulated in the vehicle, 100 mM Tris/150 mM mannitol,pH 7.4 The treatments were administered subcutaneously twice daily for14 days, whilst the animals were maintained on a high fat diet. The bodyweight of the animals was monitored daily throughout the dosing period.At day 14, blood samples for the measurement of plasma glucose andinsulin from conscious mice were obtained after a 4-hour fasting period.Mice were then anaesthetized using isoflourane and terminal blood wascollected from the capillary bed behind the eye. The followingparameters were measured: blood chemistry measurements of triglycerides,total cholesterol, non-esterified fatty acids (NEFA),beta-hydroxybutyrate and fibroblast growth factor 21 (FGF21) (Tables 14and 15 below).

The effect of treatment with liraglutide and the GLP-1/glucagon agonistpeptides G730, G797 and G812 on body weight, in comparison toliraglutide and vehicle, is shown in FIGS. 1-4. Animals treated witheither G730 or G797 showed dose dependent and continuous weight lossover the 14 day dosing period. At 50 nmol/kg, animals treated with G730and G797 experienced an about 24% change in weight at day 14 as comparedto the vehicle-treated animals.

Mice treated with G730 or G797 showed a, dose-dependent reduction inglucose levels at day 14 (Table 13). Reduced insulin levels were alsoobserved, with these two treatments, especially at the higher doses(Table 13). The insulin sensitivity index Homeostatic model assessment(HOMA) significantly improved at 20 nmol/kg G730 and 20 and 50 nmol/kgG797. HOMA is a modeling method that uses the sum of plasma insulin andglucose levels to assess β-cell function and insulin resistance (Table14). Total plasma cholesterol was lowered both by liraglutide, G730 andG797 at all doses, with less pronounced changes in plasma non-esterifiedfatty acids (NEFA) levels and plasma and hepatic triglycerides (TG).Beta-hydroxybutyrate (BeHy) had tendencies towards increased levels, inline with the body weight loss. Fibroblast growth factor 21 (FGF21)generally increased with dual GLP-1/glucagon agonist peptide treatment.

TABLE 13 Effect of GLP-1/glucagon agonist peptide treatment on glucose,insulin, and HOMA BW day 14 start (% change dose bw of vehicle GlucoseInsulin Peptide (nmol/kg) (g) SEM mean) SEM (mM) SEM (nM) SEM HOMA SEMvehicle 0 47.4 ± 3.7 0.0 ± 0 8.8 ± 0.6 0.8 ± 0.23 7.2 ± 2.0 Liraglutide27 47.5 ± 1.8 −13.3 ± 1.4 8.0 ± 0.2 0.3 ± 0.12 2.8 ± 1.1 G730 10 44.5 ±2.2 −7.5 ± 1.1 7.2 ± 0.3 * 0.4 ± 0.14 3.3 ± 1.1 G730 20 45.9 ± 3.6 −15.6± 2.2 6.7 ± 0.6 * 0.2 ± 0.06 1.7 ± 0.5 * G730 50 46.1 ± 2.4 −24.0 ± 5.15.9 ± 0.7 * 0.3 ± 0.13 2.1 ± 1.0 G797 5 47.5 ± 1.2 −5.7 ± 3.2 7.5 ± 0.30.7 ± 0.25 5.3 ± 2.0 G797 20 47.4 ± 2.2 −16.0 ± 4.4 7.1 ± 0.6 0.3 ± 0.092.0 ± 0.8 * G797 50 47.2 ± 1.8 −25.4 ± 2.0 6.6 ± 0.5 * 0.1 ± 0.01 * 0.60.1 * G812 20 49.2 ± 3.4 −8.7 ± 1.4 8.0 ± 0.4 0.7 ± 0.23 6.0 ± 2.1Results evaluated by a two-tailed distribution, two-sample unequalvariance ttest; * indicates p < 0.05 compared to vehicle.

TABLE 14 Effect of GLP-1/glucagon agonist peptide treatment onadditional blood chemistry measurements Hepatic TG Plasma Dose (g TG/100g Plasma TG NEFA Peptide (nmol/kg) Tissue) SEM (nM) SEM (nM) SEM Vehicle0 13.6 ± 0.5 0.19 ± 0.02 0.22 ± 0.01 Liraglutide 27 13.1 ± 2.1 0.24 ±0.01 0.24 ± 0.01 G730 10 9.0 ± 0.9 * 0.21 ± 0.02 0.28 ± 0.02 * G730 2017.7 ± 3.4 0.20 ± 0.03 0.26 ± 0.03 G730 50 28.1 ± 10.1 0.23 ± 0.03 0.32± 0.05 G797 5 13.0 ± 1.1 0.16 ± 0.02 0.24 ± 0.03 G797 20 17.7 ± 5.7 0.14± 0.02 0.27 ± 0.05 G797 50 15.6 ± 5.8 0.12 ± 0.02 * 0.24 ± 0.02 G812 207.9 ± 0.6 * 0.13 ± 0.01 * 0.21 ± 0.01 Plasma Dose Cholesterol FGF21Peptide (nmol/kg) (nM) SEM BeHy (umol/L) SEM (pg/mL) SEM Vehicle 0 4.65± 0.12 389 ± 46 2757 ± 317 Liraglutide 27 3.75 ± 0.16 * 345 ± 21 2481 ±650 G730 10 3.10 ± 0.16 * 428 ± 54 1963 ± 219 G730 20 2.45 ± 0.30 * 750± 318 2236 ± 300 G730 50 2.19 ± 0.23 * 1477 ± 479 5294 ± 2307 G797 53.32 ± 0.38 * 392 ± 111 2362 ± 342 G797 20 2.44 ± 0.27 * 659 ± 240 7277± 2455 G797 50 1.85 ± 0.07 * 1257 ± 285 5373 ± 813 * G812 20 2.79 ±0.24 * 333 ± 63 3207 ± 388 Results evaluated by a two-taileddistribution, two-sample unequal variance ttest; * indicates p < 0.05compared to vehicle.

G865, G933, and G796 (Study B).

A further set of GLP-1/glucagon peptides was tested in a diet inducedobesity model using the same protocol above, but with the treatmentgroups and dosing shown in Table 15:

TABLE 15 Treatment Groups for Study B Peptide Dose # of Animals VehicleNA 6 Liraglutide 26.6 nmol/kg   6 G865  5 nmol/kg 6 G865 10 nmol/kg 6G933  5 nmol/kg 6 G933 10 nmol/kg 6 G796 20 nmol/kg 6 G796 50 nmol/kg 6

GLP-1/glucagon agonist peptides G865, G933, and G796, as well asliraglutide were formulated in the vehicle, 100 mM Tris/150 mM mannitol,pH 7.4 The treatments were administered subcutaneously twice daily for14 days, whilst the animals were maintained on a high fat diet. The bodyweight of the animals was monitored daily throughout the dosing periodAt day 14, blood samples for the measurement of plasma glucose andinsulin from conscious mice were obtained after a 4-hour fasting period.Mice were then anaesthetized using isoflourane and terminal blood wascollected from the capillary bed behind the eye. The followingparameters were measured: blood chemistry measurements of triglycerides,total cholesterol, non-esterified fatty acids (NEFA),beta-hydroxybutyrate and fibroblast growth factor 21 (FGF21) (Table 16and Table 17 below).

The effect of treatment with liraglutide and the GLP-1/glucagon agonistpeptides G933, G865, G796 on body weight, in comparison to liraglutideand vehicle, is shown in FIGS. 5-8 Animals treated with either G933,G865 or G796 showed dose dependent and continuous weight loss over the14 day dosing period.

Glucose levels, insulin levels and HOMA at day 14 post-treatment areshown in Table 16. Total plasma cholesterol levels, plasmanon-esterified fatty acids (NEFA) levels, plasma and hepatictriglyceride (TG) levels, beta-hydroxy butyrate (BeHy) levels, andfibroblast growth factor 21 (FGF21) levels at day 14 post-treatment areshown in Table 17.

TABLE 16 Effect of GLP-1/glucagon agonist peptide treatment on glucose,insulin, and HOMA BW day 14 start (% change dose bw of vehicle GlucoseInsulin Peptide (nmol/kg) (g) SEM mean) SEM (mM) SEM (nM) SEM HOMA SEMvehicle 0 46.9 ± 1 0 ± 0 8.7 ± 0.8 0.58 ± 0.09 5.05 ± 0.8 Liraglutide 2746.3 ± 1.7 −14 ± 2.1 7.7 ± 0.7 0.31 ± 0.07 * 2.51 ± 0.7 * G865 5 46.9 ±0.8 −4 ± 0.1 6.2 ± 0.6 * 0.33 ± 0.08 2.14 ± 0.6 * G865 10 47.0 ± 0.9 −14± 3.4 6.6 ± 0.5 * 0.36 ± 0.06 2.43 ± 0.5 * G933 5 48.1 ± 1.6 −11 ± 2.76.2 ± 0.8 * 0.53 ± 0.13 3.31 ± 0.8 G933 10 48.6 ± 0.5 −19 ± 3.5 7.2 ±0.6 * 0.27 ± 0.07 * 1.98 ± 0.6 * G796 20 50.9 ± 1.3 −16 ± 0.6 6.1 ±0.2 * 0.38 ± 0.05 2.24 ± 0.2 * G796 50 49.7 ± 0.8 −23 ± 1.6 6.4 ± 1.1 *0.43 ± 0.14 2.87 ± 1.1 Results evaluated by a two-tailed distribution,two-sample unequal variance ttest; * indicates p < 0.05 compared tovehicle.

TABLE 17 Effect of GLP-1/glucagon agonist peptide treatment onadditional blood chemistry measurements Hepatic TG Plasma dose (g TG/100g Plasma TG NEFA Peptide (nmol/kg) tissue) SEM (mM) SEM (mM) SEM vehicle0 17.53 ± 1.30 0.25 ± 0.01 0.28 ± 0.03 Liraglutide 27 18.4 ± 2.5 0.28 ±0.03 * 0.29 ± 0.02 G865 5 20.7 ± 5.6 0.26 ± 0.03 0.29 ± 0.05 G865 1022.3 ± 5.1 0.23 ± 0.02 0.27 ± 0.03 G933 5 11.3 ± 0.8 * 0.19 ± 0.01 *0.28 ± 0.03 G933 10 14.7 ± 4.1 0.16 ± 0.01 * 0.27 ± 0.03 G796 20 9.6 ±0.9 * 0.26 ± 0.05 0.24 ± 0.02 * G796 50 9.9 ± 0.6 * 0.16 ± 0.01 * 0.21 ±0.02 Plasma dose Cholesterol BeHy Peptide (nmol/kg) (mM) SEM (umol/l)SEM FGF21 (pg/mL) SEM vehicle 0 4.56 ± 0.33 387.52 ± 87.4 2002 ± 174Liraglutide 27 3.26 ± 0.23 * 572.25 ± 82.4 * 2990 ± 729 G865 5 3.06 ±0.14 * 775.06 ± 295.5 * 8151 ± 4788 G865 10 2.89 ± 0.24 * 567.46 ±169.3 * 5953 ± 3409 G933 5 2.88 ± 0.28 * 673.08 ± 117.2 2682 ± 248 G93310 2.32 ± 0.20 * 693.56 ± 158.3 * 4459 ± 1249 G796 20 2.11 ± 0.07 *360.49 ± 51.1 6441 ± 1784 G796 50 1.91 ± 0.05 * 451.80 ± 63.4 9830 ±3278 Results evaluated by a two-tailed distribution, two-sample unequalvariance ttest; * indicates p < 0.05 compared to vehicle.

The disclosure is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the disclosure, and any compositions or methods which arefunctionally equivalent are within the scope of this disclosure. Indeed,various modifications of the disclosure in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. An isolated peptide comprising the amino acidsequence: HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30(SEQ ID NO:4) wherein, (1) X2 is S, X10 is Y, X12 is K, X13 is K, X15 isD, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X23 is V, X24 is A,X27 is V, X28 is A, and X30 is G (SEQ ID NO: 16); (2) X2 is S, X10 is K,X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E, X18 is R, X20 is R,X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, and X30 is G (SEQ IDNO: 17); (3) X2 is S, X10 is K, X12 is K, X13 is Y, X15 is E, X16 is G,X17 is Q, X18 is A, X20 is K, X21 is E, X23 is I, X24 is A, X27 is E,X28 is K, and X30 is R (SEQ ID NO: 22); (4) X2 is S, X10 is K, X12 is S,X13 is Y, X15 is D, X16 is S, X17 is R, X18 is S, X20 is R, X21 is D,X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 20);(5) X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, X17 is E,X18 is R, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A,and X30 is G (SEQ ID NO: 12); and (6) X2 is S, X10 is K, X12 is S, X13is Y, X15 is D, X16 is S, X17 is R, X18 is R, X20 is R, X21 is D, X23 isV, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO:21).
 2. Thepeptide of claim 1, further comprising a heterologous moiety associatedwith the peptide.
 3. A pharmaceutical composition comprising the peptideof claim 1, and a carrier.
 4. A kit comprising the composition of claim3.
 5. The peptide of claim 1, further comprising a modification to anamino acid.
 6. The peptide of claim 5, wherein the modification is theaddition of an acyl moiety.
 7. The peptide of claim 6, wherein themodification is a palmitoyl moiety on the N(epsilon) group of a lysineresidue.
 8. The peptide of claim 7, wherein the palmitoyl group islinked to the lysine via a linker.
 9. The peptide of claim 8, whereinthe linker is gamma glutamic acid.
 10. An isolated peptide comprisingthe amino acid sequence: HSQGTFTSDKSEYLDSERARDFVAWLEAGG (SEQ ID NO: 12).11. The peptide of claim 10, wherein the carboxyl group of the terminalglycine is the unmodified acid.
 12. The peptide of claim 11, furthercomprising a modification to an amino acid.
 13. The peptide of claim 12,wherein the modification is the addition of an acyl moiety.
 14. Thepeptide of claim 13, wherein the modification is a palmitoyl moiety onthe N(epsilon) group of a lysine residue.
 15. The peptide of claim 14,wherein the palmitoyl group is linked to the lysine via a linker. 16.The peptide of claim 15, wherein the linker is gamma glutamic acid. 17.The peptide of claim 16, wherein the peptide isHSQGTFTSDK(gammaE-palmitoyl)SEYLDSERARDFVAWLEAGG-acid.
 18. Apharmaceutical composition comprising the peptide of claim 10, and acarrier.
 19. A kit comprising the composition of claim 18.